Bicyclic compounds as modulators of androgen receptor function and method

ABSTRACT

There are disclosed bicyclic compounds according to formula I, pharmaceutical compositions containing such compounds and methods of using such compounds in the treatment of androgen receptor-associated conditions, such as age-related diseases, for example sarcopenia, 
                         
wherein R 1 , R 2 , R 5 , X and n are defined herein.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/070,025,filed Mar. 2, 2005, and as such claims priority benefit under Title 35§119(e) of U.S. Provisional Application No. 60/550,155, filed Mar. 4,2004, the contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to bicyclic compounds, methods of usingsuch compounds in the treatment of androgen receptor-associatedconditions, such as age-related diseases, for example sarcopenia, topharmaceutical compositions containing such compounds.

BACKGROUND OF THE INVENTION

Nuclear hormone receptors (NHR's) constitute a large super-family ofstructurally-related and sequence-specific gene regulators scientistshave named “ligand-dependent transcription factors.” R. M. Evans,Science, 240:889 (1988). The steroid binding NHR's (SB-NHR's) form arecognized subset of the NHR's, including the progesterone receptor(PR), androgen receptor (AR), estrogen receptor (ER), glucocorticoidreceptor (GR) and mineralocorticoid receptor (MR). The conventionalnuclear hormone receptors are generally transactivators in the presenceof ligand, which selectively bind to the NHR in a way that effects genetranscription. In the absence of a corresponding ligand, some of theorphan receptors behave as if they are transcriptionally inert. Others,however, behave as either constitutive activators or repressors. Theseorphan nuclear hormone receptors are either under the control ofubiquitous ligands that have not been identified, or do not need to bindligand to exert these activities.

The AR is a ligand-activated transcriptional regulatory protein thatmediates induction of male sexual development and function through itsactivity with endogenous androgens. In addition, androgens areassociated with male and female maintenance of muscle mass and strength,bone mass and erythropoiesis. Androgens, such as testosterone, also playan important role in many physiological processes, such asdifferentiation of male internal and external genitalia, development andmaintenance of male secondary sexual characteristics (e.g., thedevelopment of prostate, seminal vesicles, penis, scrotum, skeletalmuscle, redistribution of body fat, stimulation of long bone growth,closure of epiphyses, development of male hair growth pattern andenlargement of larynx), the maintenance of sexual behavior and function(e.g., libido and potency) and spermatogenesis (in man).

As one ages, the serum androgen concentration in the body declines. Theage dependent decline in androgens is associated with changes in bodycomposition for men and women, such as a lower percentage of muscle massand an increase in body fat, e.g., sarcopenia. In this regard,modulation of the AR gene can have an impact on the physiologicaleffects associated with androgen production. However, the effectivenessof known modulators of steroid receptors is often tempered by theirundesired side-effect profile, particularly during long-termadministration. For example, the administration of synthetic androgenshas been associated with liver damage, prostate cancer, adverse effectson male sexual function and adverse effects associated withcardiovascular and erythropoietic function.

Numerous synthetically-derived steroidal and non-steroidal agonists andantagonists have been described for the members of the SB-NHR family.Many of these agonist and antagonist ligands are used clinically in manto treat a variety of medical conditions. RU486 (mifepristone) is anexample of a synthetic antagonist of the PR, which is utilized as abirth control agent (Vegeto et al., Cell 69: 703-713 (1992)). Flutamideis an example of an antagonist of the AR, which is utilized for thetreatment of prostate cancer (Neri et al, Endo. 91, 427-437 (1972)).Tamoxifen is an example of a tissue-selective modulator of the ERfunction, that is used in the treatment of breast cancer (Smigel J.Natl. Cancer Inst. 90, 647-648 (1998)). Tamoxifen can function as anantagonist of the ER in breast tissue while acting as an agonist of theER in bone (Grese et al., Proc. Natl. Acad. Sci. USA 94, 14105-14110(1997)). Because of the tissue-selective effects seen for Tamoxifen,this agent, and agents like it, are referred to as tissue-selectiveestrogen receptor modulators. In addition to synthetically-derivednon-endogenous ligands, non-endogenous ligands for NHR's can be obtainedfrom food sources (Regal et al., Proc. Soc. Exp. Biol. Med. 223, 372-378(2000) and Hempstock et al., J. Med. Food 2, 267-269 (1999)). Theflavanoid phytoestrogens are an example of an unnatural ligand forSB-NHR's that are readily obtained from a food source such as soy(Quella et al., J. Clin. Oncol. 18, 1068-1074 (2000) and Banz et al., J.Med. Food 2, 271-273 (1999)). The ability to modulate thetranscriptional activity of an individual NHR by the addition of a smallmolecule ligand, makes these receptors ideal targets for the developmentof pharmaceutical agents for a variety of disease states.

As mentioned above, non-natural ligands can be synthetically engineeredto serve as modulators of the function of NHR's. In the case ofSB-NHR's, engineering of an unnatural ligand can include theidentification of a core structure which mimics the natural steroid coresystem. This can be achieved by random screening against severalSB-NHR's, or through directed approaches using the available crystalstructures of a variety of NHR ligand binding domains (Bourguet et al.,Nature 375, 377-382 (1995), Brzozowski, et al., Nature 389, 753-758(1997), Shiau et al., Cell 95, 927-937 (1998) and Tanenbaum et al.,Proc. Natl. Acad. Sci. USA 95, 5998-6003 (1998)). Differentialsubstitution about such a steroid mimic core can provide agents withselectivity for one receptor versus another. In addition, suchmodifications can be employed to obtain agents with agonist orantagonist activity for a particular SB-NHR. Differential substitutionabout the steroid mimic core can result in the formation of a series ofhigh affinity agonists and antagonists with specificity for, forexample, ER versus PR versus AR versus GR versus MR. Such an approach ofdifferential substitution has been reported, for example, for quinolinebased modulators of steroid NHRs in Hamann et. al., J. Med. Chem., 41,623 (1998); Hamann et. al., J. Med. Chem. 42, 210 (1999); WO 9749709;U.S. Pat. No. 5,696,133; U.S. Pat. No. 5,696,130; U.S. Pat. No.5,696,127; U.S. Pat. No. 5,693,647; U.S. Pat. No. 5,693,646; U.S. Pat.No. 5,688,810; U.S. Pat. No. 5,688,808 and WO 9619458, all incorporatedherein by reference.

Accordingly, identification of compounds which have good specificity forone or more steroid receptors, but which have reduced or nocross-reactivity for other steroid or intracellular receptors, would beof significant value in the treatment of male and femalehormone-responsive diseases. There is, therefore, a need in the art forthe identification of selective modulators of the steroid bindingnuclear hormone receptors, particularly non-steroidal, non-toxic tissueselective androgen receptor modulators, which activate the androgenreceptor in skeletal muscle while demonstrating limited or neutraleffect on other androgen responsive (e.g., prostate) tissues.

SUMMARY OF THE INVENTION

In a first embodiment of the present invention, compounds are providedwhich are capable of modulating the function of a nuclear hormonereceptor having the general formula I:

wherein

R₁ is independently selected from the group consisting of H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, arylalkyl, substitutedarylalkyl, CO₂R₄, CONR₄R₄, and CH₂OR₄;

R₂ is independently selected from the group consisting of H, OR₃, SR₃,halo, NHR₃, NHCOR₄, NHCO₂R₄, NHCONR₄R₄ and NHSO₂R₄;

R₃ is independently selected from the group consisting of H, alkyl,substituted alkyl, CHF₂, CF₃ and COR₄;

R₄, is independently selected from the group consisting of H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substitutedarylalkyl, aryl, substituted aryl, heteroaryl and substitutedheteroaryl;

R₅ is independently selected from the group consisting of H, CF₃, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substitutedarylalkyl, aryl, substituted aryl, heteroaryl and substitutedheteroaryl;

R₆ is selected from the group consisting of G and CH₂G;

G is selected from the group consisting of aryl, heterocyclo andheteroaryl, wherein said aryl, heterocyclo or heteroaryl is mono- orpolycyclic, and is optionally substituted with one or more substituentsselected from the group consisting of hydrogen, halo, CN, CF₃, OR₄,CO₂R₄, NR₄R₄, CONR₄R₄, CH₂OR₄, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, arylalkyl, substituted arylalkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl; and

n is 1 or 2; wherein

the definition of formula I above includes all prodrug esters,stereoisomers and pharmaceutically acceptable salts of formula I.

A preferred embodiment of the present invention provides compounds offormula I wherein

R₁ is H or alkyl;

R₂ is OH;

R₅ is alkyl or substituted alkyl;

X is

(where N is attached to the carbon atom bearing the R₅ substituents);and

n is 1.

A further preferred embodiment includes compounds of formula I where Gis selected from:

wherein

R₈, R₉, R₁₀ and R₁₁ are independently selected from the group consistingof H, NO₂, CN, CF₃, OR₄, CO₂R₄, NR₄R₄, CONR₄R₄, CH₂OR₄, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substitutedarylalkyl, aryl, substituted aryl, heteroaryl and substitutedheteroaryl;

A, B, C, D, E and F are independently selected from the group consistingof N and CR₁₂;

J, K, L, P and Q are independently selected from the group consisting ofNR₁₃, O, S, SO, SO₂ and CR₁₃R₁₃;

R₁₂ is independently selected from the group consisting of a direct bondand

R₁₃;

R₁₃ is independently selected from the group consisting of H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, arylalkyl, substitutedarylalkyl, CO₂R₄, CONR₄R₄ and CH₂OR₄; and

z is 0or 1.

A still further preferred embodiment of the present invention providescompounds wherein R₈ is CN.

The compounds of formula I modulate the function of the nuclear hormonereceptors, particularly the androgen receptor, and include compoundswhich are, for example, selective agonists, partial agonists,antagonists or partial antagonists of the androgen receptor. Preferablythe compounds of formula I possess activity as agonists of the androgenreceptor and may be used in the treatment of diseases or disordersassociated with androgen receptor activity, such as maintenance ofmuscle strength and function (e.g., in the elderly); reversal orprevention of frailty or age-related functional decline (“ARFD”) in theelderly (e.g., sarcopenia); prevention of catabolic side effects ofglucocorticoids; prevention and treatment of reduced bone density orgrowth (e.g., osteoporosis and osteopenia); treatment of chronic fatiguesyndrome (CFS); chronic myalgia; treatment of acute fatigue syndrome andmuscle loss following elective surgery (e.g., post-surgicalrehabilitation); treatment of urinary incontinence, acceleration ofwound healing; accelerating bone fracture repair (such as acceleratingthe recovery of hip fracture patients); treatment of wasting secondaryto fractures and wasting in connection with chronic obstructivepulmonary disease (COPD), chronic liver disease, AIDS, weightlessness,cancer cachexia, burn and trauma recovery, chronic catabolic state(e.g., coma), eating disorders (e.g., anorexia) and chemotherapy.

In a second embodiment of the present invention, pharmaceuticalcompositions, comprising at least one compound according to formula Iand at least one pharmaceutically acceptable diluent or carrier areprovided.

In a third embodiment of the present invention, methods for preventing,inhibiting or treating the progression or onset of diseases or disordersassociated with nuclear hormone receptors, comprising administering to apatient in need a therapeutically effective amount of a compoundaccording to formula I are provided.

In a preferred embodiment of the present invention, methods areprovided, wherein the nuclear hormone receptor is the androgen receptor.

In a further preferred embodiment of the present invention, methods areprovided wherein the disease or disorder associated with the androgenreceptor is selected from the group consisting of maintenance of musclestrength and function; reversal or prevention of frailty or age-relatedfunctional decline (“ARFD”) in the elderly (e.g., sarcopenia);prevention of catabolic side effects of glucocorticoids; prevention andtreatment of reduced bone density or growth (e.g., osteoporosis andosteopenia); treatment of chronic fatigue syndrome (CFS); chronicmyalgia; treatment of acute fatigue syndrome and muscle loss followingelective surgery (e.g., post-surgical rehabilitation); treatment ofurinary incontinence, acceleration of wound healing; accelerating bonefracture repair (such as accelerating the recovery of hip fracturepatients); treatment of wasting secondary to fractures and wasting inconnection with chronic obstructive pulmonary disease (COPD), chronicliver disease, AIDS, weightlessness, cancer cachexia, burn and traumarecovery, chronic catabolic state (e.g., coma), eating disorders (e.g.,anorexia) and chemotherapy.

In a still further preferred embodiment of the present invention,methods further comprising a therapeutically effective amount of atleast one other therapeutic agent are provided.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

The following abbreviations are employed herein:

-   Chiralpak®=Trademark of Chiral Technologies, Inc. Eaton, Pa.-   AcOH=acetic acid-   DBU=1,8-diazabicyclo[5.4.0]undec-7-ene-   EtOAc=ethyl acetate-   HPLC=high performance liquid chromatography-   MeOH=methanol-   MS or Mass Spec=mass spectrometry-   YMC®=trademark of YMC Co, Ltd., Kyoto, Japan-   CH₂Cl₂=dichloromethane-   CuBr=copper(I) bromide-   CuI=copper(I) iodide-   CuCN=copper(I) cyanide-   DMF=N,N-dimethylforamide-   Et₃N=triethylamine-   LDA=lithium diisopropylamide-   KOH=potassium hydroxide-   NaBH₄=sodium borohydride-   Na₂SO₄=sodium sulfate-   Pd/C=palladium on activated charcoal-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran-   mp.=melting point-   min=minute(s)-   h=hour(s)-   L=liter-   mL=milliliter-   μL=microliter-   g=gram(s)-   mg=milligram(s)-   mol=moles-   mmol=millimole(s)-   nM=nanomolar-   RT=room temperature

DEFINITIONS

The following definitions apply to the terms as used throughout thisspecification, unless otherwise limited in specific instances.

As used herein, the term “alkyl” denotes branched or unbranchedhydrocarbon chains, preferably having about 1 to about 8 carbons, suchas, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, 2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl”includes an alkyl group optionally substituted with one or morefunctional groups which are attached commonly to such chains, such as,hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano,alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl,alkenyl, nitro, amino, alkoxyl, amido, and the like to form alkyl groupssuch as trifluoro methyl, 3-hydroxyhexyl, 2-carboxypropyl,2-fluoroethyl, carboxymethyl, cyanobutyl and the like.

Unless otherwise indicated, the term “cycloalkyl” as employed hereinalone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups containing 1 to 3 rings, including monocyclicalkyl, bicyclicalkyland tricyclicalkyl, containing a total of 3 to 20 carbons forming therings, preferably 3 to 10 carbons, forming the ring and which may befused to 1 or 2 aromatic rings as described for aryl, which includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl and cyclododecyl, cyclohexenyl,

“Substituted cycloalkyl” includes a cycloalkyl group optionallysubstituted with 1 or more substituents such as halogen, alkyl, alkoxy,hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, alkylamido,alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thioland/or alkylthio and/or any of the substituents included in thedefinition of “substituted alkyl.”

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, preferably 2 to 12 carbons, and morepreferably 2 to 8 carbons in the normal chain, which include one or moredouble bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl,2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl,3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl,4-dodecenyl, 4,8,12-tetradecatrienyl, and the like. “Substitutedalkenyl” includes an alkenyl group optionally substituted with one ormore substituents, such as the substituents included above in thedefinition of “substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, preferably 2 to 12 carbons and morepreferably 2 to 8 carbons in the normal chain, which include one or moretriple bonds in the normal chain, such as 2-propynyl, 3-butynyl,2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl,3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl,3-undecynyl,4-dodecynyl and the like. “Substituted alkynyl” includes an alkynylgroup optionally substituted with one or more substituents, such as thesubstituents included above in the definition of “substituted alkyl” and“substituted cycloalkyl.”

The terms “arylalkyl”, “arylalkenyl” and “arylalkynyl” as used alone oras part of another group refer to alkyl, alkenyl and alkynyl groups asdescribed above having an aryl substituent. Representative examples ofarylalkyl include, but are not limited to, benzyl, 2-phenylethyl,3-phenylpropyl, phenethyl, benzhydryl and naphthylmethyl and the like.“Substituted arylalkyl” includes arylalkyl groups wherein the arylportion is optionally substituted with one or more substituents, such asthe substituents included above in the definition of “substituted alkyl”and “substituted cycloalkyl.”

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine.

Unless otherwise indicated, the term “aryl” or “Ar” as employed hereinalone or as part of another group refers to monocyclic and polycyclicaromatic groups containing 6 to 10 carbons in the ring portion (such asphenyl or naphthyl including 1-naphthyl and 2-naphthyl) and mayoptionally include one to three additional rings fused to a carbocyclicring or a heterocyclic ring (such as aryl, cycloalkyl, heteroaryl orcycloheteroalkyl rings), for example

“Substituted aryl” includes an aryl group optionally substituted withone or more functional groups, such as halo, haloalkyl, alkyl,haloalkyl, alkoxy, haloalkoxy, alkenyl, trifluoromethyl,trifluoromethoxy, alkynyl, cycloalkyl-alkyl, cycloheteroalkyl,cycloheteroalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy,aryloxyalkyl, arylalkoxy, alkoxycarbonyl, arylcarbonyl, arylalkenyl,aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl,heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro,cyano, amino, substituted amino wherein the amino includes 1 or 2substituents (which are alkyl, aryl or any of the other aryl compoundsmentioned in the definitions), thiol, alkylthio, arylthio,heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl,arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino,arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino orarylsulfonaminocarbonyl and/or any of the alkyl substituents set outherein.

Unless otherwise indicated, the term “heteroaryl” as used herein aloneor as part of another group refers to a 5- or 7-membered aromatic ringwhich includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygen orsulfur and such rings fused to an aryl, cycloalkyl, heteroaryl orheterocycloalkyl ring (e.g. benzothiophenyl, indolyl), and includespossible N-oxides. “Substituted heteroaryl” includes a heteroaryl groupoptionally substituted with 1 to 4 substituents, such as thesubstituents included above in the definition of “substituted alkyl” and“substituted cycloalkyl.” Examples of heteroaryl groups include thefollowing:

and the like.

The term “heterocyclo”, heterocycle or heterocyclic ring, as usedherein, represents an unsubstituted or substituted stable 5- to7-membered monocyclic ring system which may be saturated or unsaturated,and which consists of carbon atoms and from one to four heteroatomsselected from N, O or S, and wherein the nitrogen and sulfur heteroatomsmay optionally be oxidized, and the nitrogen heteroatom may optionallybe quaternized. The heterocyclic ring may be attached at any heteroatomor carbon atom which results in the creation of a stable structure.Examples of such heterocyclic groups include, but is not limited to,piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, andoxadiazolyl.

The compounds of formula I can be present as salts, which are alsowithin the scope of this invention. Pharmaceutically acceptable (i.e.,non-toxic, physiologically acceptable) salts are preferred. If thecompounds of formula I have, for example, at least one basic center,they can form acid addition salts. These are formed, for example, withstrong inorganic acids, such as mineral acids, for example sulfuricacid, phosphoric acid or a hydrohalic acid, with strong organiccarboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atomswhich are unsubstituted or substituted, for example, by halogen, forexample acetic acid, such as saturated or unsaturated dicarboxylicacids, for example oxalic, malonic, succinic, maleic, fumaric, phthalicor terephthalic acid, such as hydroxycarboxylic acids, for exampleascorbic, glycolic, lactic, malic, tartaric or citric acid, such asamino acids, (for example aspartic or glutamic acid or lysine orarginine), or benzoic acid, or with organic sulfonic acids, such as(C₁-C₄) alkyl or arylsulfonic acids which are unsubstituted orsubstituted, for example by halogen, for example methyl- orp-toluene-sulfonic acid. Corresponding acid addition salts can also beformed having, if desired, an additionally present basic center. Thecompounds of formula I having at least one acid group (for example COOH)can also form salts with bases. Suitable salts with bases are, forexample, metal salts, such as alkali metal or alkaline earth metalsalts, for example sodium, potassium or magnesium salts, or salts withammonia or an organic amine, such as morpholine, thiomorpholine,piperidine, pyrrolidine, a mono, di or tri-lower alkylamine, for exampleethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl ordimethyl-propylamine, or a mono, di or trihydroxy lower alkylamine, forexample mono, di or triethanolamine. Corresponding internal salts mayfurthermore be formed. Salts which are unsuitable for pharmaceuticaluses but which can be employed, for example, for the isolation orpurification of free compounds of formula I or their pharmaceuticallyacceptable salts, are also included.

Preferred salts of the compounds of formula I which contain a basicgroup include monohydrochloride, hydrogensulfate, methanesulfonate,phosphate or nitrate.

Preferred salts of the compounds of formula I which contain an acidgroup include sodium, potassium and magnesium salts and pharmaceuticallyacceptable organic amines.

The term “modulator” refers to a chemical compound with capacity toeither enhance (e.g., “agonist” activity) or inhibit (e.g., “antagonist”activity) a functional property of biological activity or process (e.g.,enzyme activity or receptor binding); such enhancement or inhibition maybe contingent on the occurrence of a specific event, such as activationof a signal transduction pathway, and/or may be manifest only inparticular cell types.

The term “prodrug esters” as employed herein includes esters andcarbonates formed by reacting one or more hydroxyls of compounds offormula I with alkyl, alkoxy, or aryl substituted acylating agentsemploying procedures known to those skilled in the art to generateacetates, pivalates, methylcarbonates, benzoates and the like.

Any compound that can be converted in vivo to provide the bioactiveagent (i.e., the compound of formula I) is a prodrug within the scopeand spirit of the invention.

Various forms of prodrugs are well known in the art. A comprehensivedescription of prodrugs and prodrug derivatives are described in:

-   -   (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et        al., Ch 31, (Academic Press, 1996);    -   (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier,        1985);    -   (c) A Textbook of Drug Design and Development, P.        Krogsgaard-Larson and H. Bundgaard, eds., Ch. 5, pgs, 113-191        (Harwood Academic Publishers, 1991).        Said references are incorporated herein by reference.

An administration of a therapeutic agent of the invention includesadministration of a therapeutically effective amount of the agent of theinvention. The term “therapeutically effective amount” as used hereinrefers to an amount of a therapeutic agent to treat or prevent acondition treatable by administration of a composition of the invention.That amount is the amount sufficient to exhibit a detectable therapeuticor preventative or ameliorative effect. The effect may include, forexample, treatment or prevention of the conditions listed herein. Theprecise effective amount for a subject will depend upon the subject'ssize and health, the nature and extent of the condition being treated,recommendations of the treating physician, and the therapeutics orcombination of therapeutics selected for administration. Thus, it is notuseful to specify an exact effective amount in advance.

All stereoisomers of the compounds of the instant invention arecontemplated, either in admixture or in pure or substantially pure form.The compounds of the present invention can have asymmetric centers atany of the carbon atoms including any one of the R substituents.Consequently, compounds of formula I can exist in enantiomeric ordiastereomeric forms or in mixtures thereof. The processes forpreparation can utilize racemates, enantiomers or diastereomers asstarting materials. When diastereomeric or enantiomeric products areprepared, they can be separated by conventional methods for example,chromatographic, chiral HPLC or fractional crystallization.

The compounds of formula I of the present invention can be prepared asshown in the following reaction schemes and description thereof, as wellas relevant published literature procedures that may be used by oneskilled in the art. Exemplary reagents and procedures for thesereactions appear hereinafter and in the working Examples.

Chemistry used to synthesize preferred compounds of the generalstructure I is shown below in Scheme I-III. Additional schemes will beadded to enable the entire breadth of the genus.

Scheme I describes methods for preparing compounds of formula I (subsetstructures as compounds Ia, Ib, Ic and Id). Photo-mediated [2+2]cycloaddition of compound II and III provide the adduct IV. Hydrolysisof ketal moiety under acid condition provide ketone intermediate V. Insome cases, R₂ group in compound V can be protected by a suitableprotecting group before next step. Treatment of compound V withO-mesitylenesulfonyl-hydroxylamine VI give an oxime derivative VII whichundergo a Bechmann Rearrangement to provide compounds VIII and IX. Cu orPd catalyzed N-arylation of compounds VIII and IX with compound X or XIfollowed by deprotection step in R₂ (if needed) provide compounds Ia andIb, respectively. Alkylation of compounds VIII and IX with compound XIIfollowed by deprotection in R₂ (if needed) provide compounds Ic and Id,respectively.

Scheme II describes a method to prepare compounds of formula X and XI.Intermediates XIII are treated with tert-butylnitrite or an equivalentreagent in the presence of a halide source such as CuI or CuBr to affordan aryl or heteroaryl halide of formula X or XI. Additional methods forpreparing compounds X and XI can also be found in the literature or bereadily carried out by one skilled in the art.

Scheme III describes a method to prepare compounds of formula XII.Carbonylation of either X or XI give an ester XIV. Sodium borohydridereduction of compound XIV followed by bromination with PBr₃ couldprovide compound XII. Additional methods for preparing compounds XII canalso be found in the literature or be readily carried out by one skilledin the art.

Use and Utility

A. Utilities

The compounds of the present invention modulate the function of thenuclear hormone receptors, particularly the androgen receptor, andinclude compounds which are, for example, selective agonists, partialagonists, antagonists or partial antagonists of the androgen receptor(AR). Thus, the present compounds are useful in the treatment ofAR-associated conditions. An “AR-associated condition,” as used herein,denotes a condition or disorder which can be treated by modulating thefunction or activity of an AR in a subject, wherein treatment comprisesprevention, partial alleviation or cure of the condition or disorder.Modulation may occur locally, for example, within certain tissues of thesubject, or more extensively throughout a subject being treated for sucha condition or disorder.

The compounds of the present invention can be administered to animals,preferably humans, for the treatment of a variety of conditions anddisorders, including, but not limited to maintenance of muscle strengthand function (e.g., in the elderly); reversal or prevention of frailtyor age-related functional decline (“ARFD”) in the elderly (e.g.,sarcopenia); treatment of catabolic side effects of glucocorticoids;prevention and/or treatment of reduced bone mass, density or growth(e.g., osteoporosis and osteopenia); treatment of chronic fatiguesyndrome (CFS); chronic myalgia; treatment of acute fatigue syndrome andmuscle loss following elective surgery (e.g., post-surgicalrehabilitation); accelerating of wound healing; accelerating bonefracture repair (such as accelerating the recovery of hip fracturepatients); accelerating healing of complicated fractures, e.g.distraction osteogenesis; in joint replacement; prevention ofpost-surgical adhesion formation; acceleration of tooth repair orgrowth; maintenance of sensory function (e.g., hearing, sight,olefaction and taste); treatment of periodontal disease; treatment ofwasting secondary to fractures and wasting in connection with chronicobstructive pulmonary disease (COPD), chronic liver disease, AIDS,weightlessness, cancer cachexia, burn and trauma recovery, chroniccatabolic state (e.g., coma), eating disorders (e.g., anorexia) andchemotherapy; treatment of cardiomyopathy; treatment ofthrombocytopenia; treatment of growth retardation in connection withCrohn's disease; treatment of short bowel syndrome; treatment ofirritable bowel syndrome; treatment of inflammatory bowel disease;treatment of Crohn's disease and ulcerative colitis; treatment ofcomplications associated with transplantation; treatment ofphysiological short stature including growth hormone deficient childrenand short stature associated with chronic illness; treatment of obesityand growth retardation associated with obesity; treatment of anorexia(e.g., associated with cachexia or aging); treatment of hypercortisolismand Cushing's syndrome; Paget's disease; treatment of osteoarthritis;induction of pulsatile growth hormone release; treatment ofosteochondrodysplasias; treatment of depression, nervousness,irritability and stress; treatment of reduced mental energy and lowself-esteem (e.g., motivation/assertiveness); improvement of cognitivefunction (e.g., the treatment of dementia, including Alzheimer's diseaseand short term memory loss); treatment of catabolism in connection withpulmonary dysfunction and ventilator dependency; treatment of cardiacdysfunction (e.g., associated with valvular disease, myocardialinfarction, cardiac hypertrophy or congestive heart failure); loweringblood pressure; protection against ventricular dysfunction or preventionof reperfusion events; treatment of adults in chronic dialysis; reversalor slowing of the catabolic state of aging; attenuation or reversal ofprotein catabolic responses following trauma (e.g., reversal of thecatabolic state associated with surgery, congestive heart failure,cardiac myopathy, burns, cancer, COPD etc.); reducing cachexia andprotein loss due to chronic illness such as cancer or AIDS; treatment ofhyperinsulinemia including nesidioblastosis; treatment ofimmunosuppressed patients; treatment of wasting in connection withmultiple sclerosis or other neurodegenerative disorders; promotion ofmyelin repair; maintenance of skin thickness; treatment of metabolichomeostasis and renal homeostasis (e.g., in the frail elderly);stimulation of osteoblasts, bone remodeling and cartilage growth;regulation of food intake; treatment of insulin resistance, includingNIDDM, in mammals (e.g., humans); treatment of insulin resistance in theheart; improvement of sleep quality and correction of the relativehyposomatotropism of senescence due to high increase in REM sleep and adecrease in REM latency; treatment of hypothermia; treatment ofcongestive heart failure; treatment of lipodystrophy (e.g., in patientstaking HIV or AIDS therapies such as protease inhibitors); treatment ofmuscular atrophy (e.g., due to physical inactivity, bed rest or reducedweight-bearing conditions); treatment of musculoskeletal impairment(e.g., in the elderly); improvement of the overall pulmonary function;treatment of sleep disorders; and the treatment of the catabolic stateof prolonged critical illness; treatment of hirsutism, acne, seborrhea,androgenic alopecia, anemia, hyperpilosity, benign prostate hypertrophy,adenomas and neoplasies of the prostate (e.g., advanced metastaticprostate cancer) and malignant tumor cells containing the androgenreceptor, such as is the case for breast, brain, skin, ovarian, bladder,lymphatic, liver and kidney cancers; cancers of the skin, pancreas,endometrium, lung and colon; osteosarcoma; hypercalcemia of malignancy;metastatic bone disease; treatment of spermatogenesis, endometriosis andpolycystic ovary syndrome; conteracting preeclampsia, eclampsia ofpregnancy and preterm labor; treatment of premenstrual syndrome;treatment of vaginal dryness; age related decreased testosterone levelsin men, male menopause, hypogonadism, male hormone replacement, male andfemale sexual dysfunction (e.g., erectile dysfunction, decreased sexdrive, sexual well-being, decreased libido), urinary incontinence, maleand female contraception, hair loss, Reaven's Syndrome and theenhancement of bone and muscle performance/strength. The term treatmentis also intended to include prophylactic treatment.

In addition, the conditions, diseases, and maladies collectivelyreferenced to as “Syndrome X” or Metabolic Syndrome as detailed inJohannsson J. Clin. Endocrinol. Metab., 82, 727-34 (1997), may betreated employing the compounds of the invention.

B. Combinations

The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, a therapeuticallyeffective amount of at least one of the compounds of formula I, alone orin combination with a pharmaceutical carrier or diluent. Optionally,compounds of the present invention can be used alone, in combinationwith other compounds of the invention, or in combination with one ormore other therapeutic agent(s), e.g., an antibiotic or otherpharmaceutically active material.

The compounds of the present invention may be combined with growthpromoting agents, such as, but not limited to, TRH, diethylstilbesterol,theophylline, enkephalins, E series prostaglandins, compounds disclosedin U.S. Pat. No. 3,239,345, e.g., zeranol, and compounds disclosed inU.S. Pat. No. 4,036,979, e.g., sulbenox or peptides disclosed in U.S.Pat. No. 4,411,890.

The compounds of the invention may also be used in combination withgrowth hormone secretagogues such as GHRP-6, GHRP-1 (as described inU.S. Pat. No. 4,411,890 and publications WO 89/07110 and WO 89/07111),GHRP-2 (as described in WO 93/04081), NN703 (Novo Nordisk), LY444711(Lilly), MK-677 (Merck), CP424391 (Pfizer) and B-HT920, or with growthhormone releasing factor and its analogs or growth hormone and itsanalogs or somatomedins including IGF-1 and IGF-2, or withalpha-adrenergic agonists, such as clonidine or serotonin 5-HT_(D)agonists, such as sumatriptan, or agents which inhibit somatostatin orits release, such as physostigmine and pyridostigmine. A still furtheruse of the disclosed compounds of the invention is in combination withparathyroid hormone, PTH(1-34) or bisphosphonates, such as MK-217(alendronate).

A still further use of the compounds of the invention is in combinationwith estrogen, testosterone, a selective estrogen receptor modulator,such as tamoxifen or raloxifene, or other androgen receptor modulators,such as those disclosed in Edwards, J. P. et. al., Bio. Med. Chem. Let.,9, 1003-1008 (1999) and Hamann, L. G. et. al., J. Med. Chem., 42,210-212 (1999).

A further use of the compounds of this invention is in combination withprogesterone receptor agonists (“PRA”), such as levonorgestrel,medroxyprogesterone acetate (MPA).

The compounds of the present invention may be employed alone or incombination with each other and/or other modulators of nuclear hormonereceptors or other suitable therapeutic agents useful in the treatmentof the aforementioned disorders including: anti-diabetic agents;anti-osteoporosis agents; anti-obesity agents; anti-inflammatory agents;anti-anxiety agents; anti-depressants; anti-hypertensive agents;anti-platelet agents; anti-thrombotic and thrombolytic agents; cardiacglycosides; cholesterol/lipid lowering agents; mineralocorticoidreceptor antagonists; phosphodiesterase inhibitors; protein tyrosinekinase inhibitors; thyroid mimetics (including thyroid receptoragonists); anabolic agents; HIV or AIDS therapies; therapies useful inthe treatment of Alzheimer's disease and other cognitive disorders;therapies useful in the treatment of sleeping disorders;anti-proliferative agents; and anti-tumor agents.

Examples of suitable anti-diabetic agents for use in combination withthe compounds of the present invention include biguanides (e.g.,metformin), glucosidase inhibitors (e.g., acarbose), insulins (includinginsulin secretagogues or insulin sensitizers), meglitinides (e.g.,repaglinide), sulfonylureas (e.g., glimepiride, glyburide andglipizide), biguanide/glyburide combinations (e.g., Glucovance®),thiazolidinediones (e.g., troglitazone, rosiglitazone and pioglitazone),PPAR-alpha agonists, PPAR-gamma agonists, PPAR alpha/gamma dualagonists, SGLT2 inhibitors, glycogen phosphorylase inhibitors,inhibitors of fatty acid binding protein (aP2) such as those disclosedin U.S. Ser. No. 09/519,079 filed Mar. 6, 2000, glucagon-like peptide-1(GLP-1), and dipeptidyl peptidase IV (DPP4) inhibitors such as thosedisclosed in WO 01/68603.

Examples of suitable anti-osteoporosis agents for use in combinationwith the compounds of the present invention include alendronate,risedronate, PTH, PTH fragment, raloxifene, calcitonins, steroidal ornon-steroidal progesterone receptor agonists, RANK ligand antagonists,calcium sensing receptor antagonists, TRAP inhibitors, selectiveestrogen receptor modulators (SERM's), estrogen and AP-1 inhibitors.

Examples of suitable anti-obesity agents for use in combination with thecompounds of the present invention include aP2 inhibitors, such as thosedisclosed in U.S. Ser. No. 09/519,079 filed Mar. 6, 2000, PPAR gammaantagonists, PPAR delta agonists, beta 3 adrenergic agonists, such asAJ9677 (Takeda/Dainippon), L750355 (Merck), or CP331648 (Pfizer) orother known beta 3 agonists as disclosed in U.S. Pat. Nos. 5,541,204,5,770,615, 5,491,134, 5,776,983 and 5,488,064, a lipase inhibitor, suchas orlistat or ATL-962 (Alizyme), a serotonin (and dopamine) reuptakeinhibitor, such as sibutramine, topiramate (Johnson & Johnson) oraxokine (Regeneron), a thyroid receptor beta drug, such as a thyroidreceptor ligand as disclosed in WO 97/21993 (U. Cal SF), WO 99/00353(KaroBio) and GB98/284425 (KaroBio), and/or an anorectic agent, such asdexamphetamine, phentermine, phenylpropanolamine or mazindol.

Examples of suitable anti-inflammatory agents for use in combinationwith the compounds of the present invention include prednisone,dexamethasone, Enbrel®, cyclooxygenase inhibitors (i.e., COX-1 and/orCOX-2 inhibitors such as NSAIDs, aspirin, indomethacin, ibuprofen,piroxicam, Naproxen®, Celebrex®, Vioxx®), CTLA4-Ig agonists/antagonists,CD40 ligand antagonists, IMPDH inhibitors, such as mycophenolate(CellCept®), integrin antagonists, alpha-4 beta-7 integrin antagonists,cell adhesion inhibitors, interferon gamma antagonists, ICAM-1, tumornecrosis factor (TNF) antagonists (e.g., infliximab, OR1384),prostaglandin synthesis inhibitors, budesonide, clofazimine, CNI-1493,CD4 antagonists (e.g., priliximab), p38 mitogen-activated protein kinaseinhibitors, protein tyrosine kinase (PTK) inhibitors, IKK inhibitors,and therapies for the treatment of irritable bowel syndrome (e.g.,Zelmac® and Maxi-K® openers such as those disclosed in U.S. Pat. No.6,184,231 B1).

Examples of suitable anti-anxiety agents for use in combination with thecompounds of the present invention include diazepam, lorazepam,buspirone, oxazepam, and hydroxyzine pamoate.

Examples of suitable anti-depressants for use in combination with thecompounds of the present invention include citalopram, fluoxetine,nefazodone, sertraline, and paroxetine.

Examples of suitable anti-hypertensive agents for use in combinationwith the compounds of the present invention include beta adrenergicblockers, calcium channel blockers (L-type and T-type; e.g. diltiazem,verapamil, nifedipine, amlodipine and mybefradil), diuretics (e.g.,chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide,bendroflumethiazide, methylchlorothiazide, trichloromethiazide,polythiazide, benzthiazide, ethacrynic acid tricrynafen, chlorthalidone,furosemide, musolimine, bumetanide, triamtrenene, amiloride,spironolactone), renin inhibitors, ACE inhibitors (e.g., captopril,zofenopril, fosinopril, enalapril, ceranopril, cilazopril, delapril,pentopril, quinapril, ramipril, lisinopril), AT-1 receptor antagonists(e.g., losartan, irbesartan, valsartan), ET receptor antagonists (e.g.,sitaxsentan, atrsentan and compounds disclosed in U.S. Pat. Nos.5,612,359 and 6,043,265), Dual ET/AII antagonist (e.g., compoundsdisclosed in WO 00/01389), neutral endopeptidase (NEP) inhibitors,vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilatand gemopatrilat), and nitrates.

Examples of suitable anti-platelet agents for use in combination withthe compounds of the present invention include GPIIb/IIIa blockers(e.g., abciximab, eptifibatide, tirofiban), P2Y12 antagonists (e.g.,clopidogrel, ticlopidine, CS-747), thromboxane receptor antagonists(e.g., ifetroban), aspirin, and PDE-III inhibitors (e.g., dipyridamole)with or without aspirin.

Examples of suitable cardiac glycosides for use in combination with thecompounds of the present invention include digitalis and ouabain.

Examples of suitable cholesterol/lipid lowering agents for use incombination with the compounds of the present invention include HMG-CoAreductase inhibitors (e.g., pravastatin, lovastatin, atorvastatin,simvastatin, NK-104 (a.k.a. itavastatin, or nisvastatin or nisbastatin)and ZD4522 (a.k.a. rosuvastatin, or atavastatin or visastatin)),squalene synthetase inhibitors, fibrates, bile acid sequestrants, ACATinhibitors, MTP inhibitors, lipooxygenase inhibitors, cholesterolabsorption inhibitors, and cholesterol ester transfer protein inhibitors(e.g., CP-529414).

Examples of suitable mineralocorticoid receptor antagonists for use incombination with the compounds of the present invention includespironolactone and eplerinone.

Examples of suitable phosphodiesterase (PDE) inhibitors for use incombination with the compounds of the present invention include PDE-3inhibitors such as cilostazol, and phosphodiesterase-5 inhibitors (PDE-5inhibitors) such as sildenafil.

Examples of suitable thyroid mimetics for use in combination with thecompounds of the present invention include thyrotropin, polythyroid,KB-130015, and dronedarone.

Examples of suitable anabolic agents for use in combination with thecompounds of the present invention include testosterone, TRHdiethylstilbesterol, estrogens, β-agonists, theophylline, anabolicsteroids, dehydroepiandrosterone, enkephalins, E-series prostagladins,retinoic acid and compounds as disclosed in U.S. Pat. No. 3,239,345,e.g., Zeranol®; U.S. Pat. No. 4,036,979, e.g., Sulbenox® or peptides asdisclosed in U.S. Pat. No. 4,411,890.

Examples of suitable HIV or AIDS therapies for use in combination withthe compounds of the present invention include indinavir sulfate,saquinavir, saquinavir mesylate, ritonavir, lamivudine, zidovudine,lamivudine/zidovudine combinations, zalcitabine, didanosine, stavudine,and megestrol acetate.

Examples of suitable therapies for treatment of Alzheimer's disease andcognitive disorders for use in combination with the compounds of thepresent invention include donepezil, tacrine, revastigmine, 5HT6, gammasecretase inhibitors, beta secretase inhibitors, SK channel blockers,Maxi-K blockers, and KCNQs blockers.

Examples of suitable therapies for treatment of sleeping disorders foruse in combination with the compounds of the present invention includemelatonin analogs, melatonin receptor antagonists, ML1B agonists, andGABA/NMDA receptor antagonists.

Examples of suitable anti-proliferative agents for use in combinationwith the compounds of the present invention include cyclosporin A,paclitaxel, FK-506, and adriamycin.

Examples of suitable anti-tumor agents for use in combination with thecompounds of the present invention include paclitaxel, adriamycin,epothilones, cisplatin and carboplatin.

Compounds of the present invention may further be used in combinationwith nutritional supplements such as those described in U.S. Pat. No.5,179,080, especially in combination with whey protein or casein, aminoacids (such as leucine, branched amino acids and hydroxymethylbutyrate),triglycerides, vitamins (e.g., A, B6, B12, folate, C, D and E), minerals(e.g., selenium, magnesium, zinc, chromium, calcium and potassium),carnitine, lipoic acid, creatinine, B-hyroxy-B-methylbutyriate (Juven)and coenzyme Q-10.

In addition, compounds of the present invention may be used incombination with therapeutic agents used in the treatment of sexualdysfunction, including but not limited to PDE-5 inhibitors, such assildenafil or IC-351.

Compounds of the present invention may further be used in combinationwith antiresorptive agents, hormone replacement therapies, vitamin Danalogues, elemental calcium and calcium supplements, cathepsin Kinhibitors, MMP inhibitors, vitronectin receptor antagonists, Src SH₂antagonists, vacular —H⁺-ATPase inhibitors, ipriflavone, fluoride,Tibolone, prostanoids, 17-beta hydroxysteroid dehydrogenase inhibitorsand Src kinase inhibitors.

Compounds of the present invention may be used in combination with malecontraceptives, such as nonoxynol 9 or therapeutic agents for thetreatment of hair loss, such as minoxidil and finasteride orchemotherapeutic agents, such as with LHRH agonists.

Further, the compounds of the present invention may be used incombination with anti-cancer and cytotoxic agents, including but notlimited to alkylating agents such as nitrogen mustards, alkylsulfonates, nitrosoureas, ethylenimines, and triazenes; antimetabolitessuch as folate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5α-reductase inhibitors; inhibitors of17β-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol®), docetaxel (Taxotere®), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies. The compounds of the invention may also be used inconjunction with radiation therapy.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred member of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, caminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or porfiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents include the following:epothilone derivatives as found in German Patent No. 4138042.8; WO97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/01124, WO99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890, WO99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclin dependentkinase inhibitors as found in WO 99/24416 (see also U.S. Pat. No.6,040,321); and prenyl-protein transferase inhibitors as found in WO97/30992 and WO 98/54966; and agents such as those described genericallyand specifically in U.S. Pat. No. 6,011,029 (the compounds of which U.S.patent can be employed together with any NHR modulators (including, butnot limited to, those of present invention) such as AR modulators, ERmodulators, with LHRH modulators, or with surgical castration,especially in the treatment of cancer).

The above other therapeutic agents, when employed in combination withthe compounds of the present invention, may be used, for example, inthose amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

The compounds of the formula I can be administered for any of the usesdescribed herein by any suitable means, for example, orally, such as inthe form of tablets, capsules, granules or powders; sublingually;bucally; parenterally, such as by subcutaneous, intravenous,intramuscular, or intrasternal injection or infusion techniques (e.g.,as sterile injectable aqueous or non-aqueous solutions or suspensions);nasally, including administration to the nasal membranes, such as byinhalation spray; topically, such as in the form of a cream or ointment;or rectally such as in the form of suppositories; in dosage unitformulations containing non-toxic, pharmaceutically acceptable vehiclesor diluents. The present compounds can, for example, be administered ina form suitable for immediate release or extended release. Immediaterelease or extended release can be achieved by the use of suitablepharmaceutical compositions comprising the present compounds, or,particularly in the case of extended release, by the use of devices suchas subcutaneous implants or osmotic pumps. The present compounds canalso be administered liposomally.

Exemplary compositions for oral administration include suspensions whichcan contain, for example, microcrystalline cellulose for imparting bulk,alginic acid or sodium alginate as a suspending agent, methylcelluloseas a viscosity enhancer, and sweeteners or flavoring agents such asthose known in the art; and immediate release tablets which can contain,for example, microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and/or lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants such as those known inthe art. The compounds of formula I can also be delivered through theoral cavity by sublingual and/or buccal administration. Molded tablets,compressed tablets or freeze-dried tablets are exemplary forms which maybe used. Exemplary compositions include those formulating the presentcompound(s) with fast dissolving diluents such as mannitol, lactose,sucrose and/or cyclodextrins. Also included in such formulations may behigh molecular weight excipients such as celluloses (avicel) orpolyethylene glycols (PEG). Such formulations can also include anexcipient to aid mucosal adhesion such as hydroxy propyl cellulose(HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methylcellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agentsto control release such as polyacrylic copolymer (e.g. Carbopol 934).Lubricants, glidants, flavors, coloring agents and stabilizers may alsobe added for ease of fabrication and use.

Exemplary compositions for nasal aerosol or inhalation administrationinclude solutions in saline which can contain, for example, benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, and/or other solubilizing or dispersing agents such asthose known in the art.

Exemplary compositions for parenteral administration include injectablesolutions or suspensions which can contain, for example, suitablenon-toxic, parenterally acceptable diluents or solvents, such asmannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodiumchloride solution, or other suitable dispersing or wetting andsuspending agents, including synthetic mono- or diglycerides, and fattyacids, including oleic acid, or Cremaphor.

Exemplary compositions for rectal administration include suppositorieswhich can contain, for example, a suitable non-irritating excipient,such as cocoa butter, synthetic glyceride esters or polyethyleneglycols, which are solid at ordinary temperatures, but liquify and/ordissolve in the rectal cavity to release the drug.

Exemplary compositions for topical administration include a topicalcarrier such as Plastibase (mineral oil gelled with polyethylene).

The effective amount of a compound of the present invention can bedetermined by one of ordinary skill in the art, and includes exemplarydosage amounts for an adult human of from about 0.01 to 2000 mg ofactive compound per day, which can be administered in a single dose orin the form of individual divided doses, such as from 1 to 4 times perday. It will be understood that the specific dose level and frequency ofdosage for any particular subject can be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the species, age, body weight, general health, sex and diet of thesubject, the mode and time of administration, rate of excretion, drugcombination, and severity of the particular condition. Preferredsubjects for treatment include animals, most preferably mammalianspecies such as humans, and domestic animals such as dogs, cats and thelike, subject to NHR-associated conditions.

Transactivation Assays

AR Specific Assay

Compounds of the present invention were tested in transactivation assaysof a transfected reporter construct and using the endogenous androgenreceptor of the host cells. The transactivation assay provides a methodfor identifying functional agonists and partial agonists that mimic, orantagonists that inhibit, the effect of native hormones, in this case,dihydrotestosterone (DHT). This assay can be used to predict in vivoactivity as there is a good correlation in both series of data. See,e.g. T. Berger et al., J. Steroid Biochem. Molec. Biol. 773 (1992), thedisclosure of which is herein incorporated by reference.

For the transactivation assay a reporter plasmid is introduced bytransfection (a procedure to induce cells to take foreign genes) intothe respective cells. This reporter plasmid, comprising the cDNA for areporter protein, such as secreted alkaline phosphatase (SEAP),controlled by prostate specific antigen (PSA) upstream sequencescontaining androgen response elements (AREs). This reporter plasmidfunctions as a reporter for the transcription-modulating activity of theAR. Thus, the reporter acts as a surrogate for the products (mRNA thenprotein) normally expressed by a gene under control of the AR and itsnative hormone. In order to detect antagonists, the transactivationassay is carried out in the presence of constant concentration of thenatural AR hormone (DHT) known to induce a defined reporter signal.Increasing concentrations of a suspected antagonist will decrease thereporter signal (e.g., SEAP production). On the other hand, exposing thetransfected cells to increasing concentrations of a suspected agonistwill increase the production of the reporter signal.

For this assay, LNCaP and NDA 453 cells were obtained from the AmericanType Culture Collection (Rockville, Md.), and maintained in RPMI 1640 orDMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco)respectively. The respective cells were transiently transfected byelectroporation according to the optimized procedure described byHeiser, 130 Methods Mol. Biol., 117 (2000), with thepSEAP2/PSA540/Enhancer reporter plasmid. The reporter plasmid, wasconstructed as follows: commercial human placental genomic DNA was usedto generate by Polymerase Cycle Reaction (PCR) a fragment containing theBglII site (position 5284) and the Hind III site at position 5831 of thehuman prostate specific antigen promoter (Accession # U37672), Schuur,et al., J. Biol. Chem., 271 (12): 7043-51 (1996). This fragment wassubcloned into the pSEAP2/basic (Clontech) previously digested withBglII and HindIII to generate the pSEAP2/PSA540 construct. Then afragment bearing the fragment of human PSA upstream sequence betweenpositions -5322 and -3873 was amplified by PCR from human placentalgenomic DNA. A XhoI and a BglII sites were introduced with the primers.The resulting fragment was subcloned into pSEAP2/PSA540 digested withXhoI and BglII respectively, to generate the pSEAP2/PSA540/Enhancerconstruct. LNCaP and MDA MB-453 cells were collected in media containing10% charcoal stripped FBS. Each cell suspension was distributed into twoGene Pulser Cuvetts (Bio-Rad) which then received 8 μg of the reporterconstruct, and electoporated using a Bio-Rad Gene Pulser at 210 voltsand 960 μFaraday. Following the transfections the cells were washed andincubated with media containing charcoal stripped fetal bovine serum inthe absence (blank) or presence (control) of 1 nM dihydrotestosterone(DHT; Sigma Chemical) and in the presence or absence of the standardanti-androgen bicalutamide or compounds of the present invention inconcentrations ranging from 10⁻¹⁰ to 10⁻⁵ M (sample). Duplicates wereused for each sample. The compound dilutions were performed on a Biomek2000 laboratory workstation.

After 48 h, a fraction of the supernatant was assayed for SEAP activityusing the Phospha-Light Chemiluminescent Reporter Gene Assay System(Tropix, Inc). Viability of the remaining cells was determined using theCellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTSAssay, Promega). Briefly, a mix of a tetrazolium compound(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS) and an electron coupling reagent (phenazinemethosulfate; PMS) are added to the cells. MTS (Owen's reagent) isbioreduced by cells into a formazan that is soluble in tissue culturemedium, and therefore its absorbance at 490 nm can be measured directlyfrom 96 well assay plates without additional processing. The quantity offormazan product as measured by the amount of 490 nm absorbance isdirectly proportional to the number of living cells in culture. For eachreplicate the SEAP reading was normalized by the Abs490 value derivedfrom the MTS assay. For the antagonist mode, the % Inhibition wascalculated as:% Inhibition=100×(1−[average control−average blank/averagesample−average blank])Data was plotted and the concentration of compound that inhibited 50% ofthe normalized SEAP was quantified (IC₅₀).

For the agonist mode % Control was referred as the effect of the testedcompound compared to the maximal effect observed with the naturalhormone, in this case DHT, and was calculated as:% Control=100×average sample−average blank/average control−average blankData was plotted and the concentration of compound that activates tolevels 50% of the normalized SEAP for the control was quantified (EC₅₀).GR Specificity Assay

The reporter plasmid utilized was comprised of the cDNA for the reporterSEAP protein, as described for the AR specific transactivation assay.Expression of the reporter SEAP protein was controlled by the mousemammary tumor virus long terminal repeat (MMTV LTR) sequences thatcontains three hormone response elements (HREs) that can be regulated byboth GR and PR see, e.g. G. Chalepakis et al., Cell, 53(3), 371 (1988).This plasmid was transfected into A549 cells, which expresses endogenousGR, to obtain a GR specific transactivation assay. A549 cells wereobtained from the American Type Culture Collection (Rockville, Md.), andmaintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS;Gibco). Determination of the GR specific antagonist activity of thecompounds of the present invention was identical to that described forthe AR specific transactivation assay, except that the DHT was replacedwith 5 nM dexamethasone (Sigma Chemicals), a specific agonist for GR.Determination of the GR specific agonist activity of the compounds ofthe present invention was performed as described for the ARtransactivation assay, wherein one measures the activation of the GRspecific reporter system by the addition of a test compound, in theabsence of a known GR specific agonists ligand.

PR Specific Assay

The reporter plasmid utilized was comprised of the cDNA for the reporterSEAP protein, as described for the AR specific transactivation assay.Expression of the reporter SEAP protein was controlled by the mousemammary tumor virus long terminal repeat (MMTV LTR) sequences thatcontains three hormone response elements (HRE's) that can be regulatedby both GR and PR. This plasmid was transfected into T47D, whichexpresses endogenous PR, to obtain a PR specific transactivation assay.T47D cells were obtained from the American Type Culture Collection(Rockville, Md.), and maintained in DMEM medium supplemented with 10%fetal bovine serum (FBS; Gibco). Determination of the PR specificantagonist activity of the compounds of the present invention wasidentical to that described for the AR specific transactivation assay,except that the DHT was replaced with 1 nM Promegastone (NEN), aspecific agonist for PR. Determination of the PR specific agonistactivity of the compounds of the present invention was performed asdescribed for the AR transactivation assay, wherein one measures theactivation of the PR specific reporter system by the addition of a testcompound, in the absence of a known PR specific agonists ligand.

AR Binding Assay

For the whole-cell binding assay, human LNCAP cells (T877A mutant AR) orMDA 453 (wild type AR) in 96-well microtiter plates containing RPMI 1640or DMEM supplemented with 10% charcoal stripped CA-FBS (CocalecoBiologicals) respectively, were incubated at 37° C. to remove anyendogenous ligand that might be complexed with the receptor in thecells. After 48 h, either a saturation analysis to determine the K_(d)for tritiated dihydrotestosterone, [³H]-DHT, or a competitive bindingassay to evaluate the ability of test compounds to compete with [³H]-DHTwere performed. For the saturation analysis, media (RPMI 1640 orDMEM-0.2% CA-FBS) containing [³H]-DHT (in concentrations ranging from0.1 nM to 16 nM) in the absence (total binding) or presence(non-specific binding) of a 500-fold molar excess of unlabeled DHT wereadded to the cells. After 4 h at 37° C., an aliquot of the total bindingmedia at each concentration of [³H]-DHT was removed to estimate theamount of free [³H]-DHT. The remaining media was removed, cells werewashed three times with PBS and harvested onto UniFilter GF/B plates(Packard), Microscint (Packard) was added and plates counted in aTop-Counter (Packard) to evaluate the amount of bound [³H]-DHT.

For the saturation analysis, the difference between the total bindingand the non-specific binding, was defined as specific binding. Thespecific binding was evaluated by Scatchard analysis to determine theK_(d) for [³H]-DHT. See e.g. D. Rodbard, Mathematics and statistics ofligand assays: an illustrated guide: In: J. Langon and J. J. Clapp,eds., Ligand Assay, Masson Publishing U.S.A., Inc., New York, pp. 45-99,(1981), the disclosure of which is herein incorporated by reference.

For the competition studies, media containing 1 nM [³H]-DHT andcompounds of the invention (“test compounds”) in concentrations rangingfrom 10⁻¹⁰ to 10⁻⁵ M were added to the cells. Two replicates were usedfor each sample. After 4 h at 37° C., cells were washed, harvested andcounted as described above. The data was plotted as the amount of[³H]-DHT (% of control in the absence of test compound) remaining overthe range of the dose response curve for a given compound. Theconcentration of test compound that inhibited 50% of the amount of[³H]-DHT bound in the absence of competing ligand was quantified (IC₅₀)after log-logit transformation. The K₁ values were determined byapplication of the Cheng-Prusoff equation to the IC₅₀ values, where:

$K_{I} = \frac{{IC}_{50}}{\left( {1 + {{\left( {}^{3}{H - {D\; H\; T}} \right)/K_{d}}\mspace{14mu}{{for}\mspace{14mu}}^{3}H} - {D\; H\; T}} \right)}$After correcting for non-specific binding, IC₅₀ values were determined.The IC₅₀ is defined as the concentration of competing ligand needed toreduce specific binding by 50%. The K_(d)s for [³H]-DHT for MDA 453 andLNCaP were 0.7 and 0.2 nM respectively.C2C12 Mouse Myoblast Transactivation Assay

Two functional transactivation assays were developed to assess theefficacy of androgen agonists in a muscle cell background using aluciferase reporter. The first assay (ARTA Stable 1) uses a cell line,Stable 1 (clone #72), which expresses the full length rat androgenreceptor but requires the transient transfection of anenhancer/reporter. This cell line was derived from C2C12 mouse moyoblastcells. The second assay (ARTA Stable 2) uses a cell line, Stable 2(clone #133), derived from Stable 1 which expresses both rAR and theenhancer/luciferase reporter.

The enhancer/reporter construct used in this system ispGL3/2XDR-1/luciferase. 2×DR-1 was reported to be an AR specificresponse element in CV-1 cells, Brown et. al. The Journal of BiologicalChemistry 272, 8227-8235, (1997). It was developed by random mutagenesisof an AR/GR consensus enhancer sequence.

ARTA Stable 1

1. Stable 1 cells are plated in 96 well format at 6,000 cells/well inhigh glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029)containing 10% charcoal and dextran treated FBS (HyClone Cat. No.:SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), IX MEMNa Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5×Antibiotic-Antimycotic, and 800 μg/mL Geneticin (Gibco BRL, Cat. No.:10131-035).

2. 48 h later, cells are transfected with pGL3/2XDR-1/luciferase usingLipofectAMINE Plus™ Reagent (Gibco BRL, Cat. No.: 10964-013).Specifically, 5 ng/well pGL3/2XDR-1/luciferase DNA and 50 ng/well SalmonSperm DNA (as carrier) are diluted with 5 μl/well Opti-MEMem media(Gibco BRL, Cat. No.: 31985-070). To this, 0.5 μl/well Plus reagent isadded. This mixture is incubated for 15 min at rt. In a separate vessel,0.385 μl/well LipofectAMINE reagent is diluted with 5 μl/well Opti-MEM.The DNA mixture is then combined with the LipofectAMINE mixture andincubated for an additional 15 min at rt. During this time, the mediafrom the cells is removed and replaced with 60 μl/well of Opti-MEM. Tothis is added 10 μl/well of the DNA/LipofectAMINE transfection mixture.The cells are incubated for 4 h.

3. The transfection mixture is removed from the cells and replaced with90 μl of media as in #1 above.

4. 10 μl/well of appropriate drug dilution is placed in each well.

5. 24 h later, the Steady-Glo™ Luciferase Assay System is used to detectactivity according to the manufacturer's instructions (Promega, Cat.No.: E2520).

ARTA Stable 2

1. Stable 2 cells are plated in 96 well format at 6,000 cells/well inhigh glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029)containing 10% charcoal and dextran treated FBS (HyClone Cat. No.:SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), 1×MEMNa Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5×Antibiotic-Antimycotic, 800 μg/mL Geneticin (Gibco BRL, Cat. No.:10131-035) and 800 μg/mL Hygromycin β (Gibco BRL, Cat. No.: 10687-010).

2. 48 h later, the media on the cells is removed and replaced with 90 μlfresh. 10 μl/well of appropriate drug dilution is placed in each well.

3. 24 h later, the Steady-Glo™ Luciferase Assay System is used to detectactivity according to the manufacturer's instructions (Promega, Cat. No.E2520).

Proliferation Assays

Human Prostate Cell Proliferation Assay

Compounds of the present invention were tested (“test compounds”) on theproliferation of human prostate cancer cell lines. For that, MDA PCa2bcells, a cell line derived from the metastasis of a patient that failedcastration, Navone et al., Clin. Cancer Res., 3, 2493-500 (1997), wereincubated with or without the test compounds for 72 h and the amount of[³H]-thymidine incorporated into DNA was quantified as a way to assessnumber of cells and therefore proliferation. The MDA PCa2b cell line wasmaintained in BRFF—HPC1 media (Biological Research Faculty & FacilityInc., MD) supplemented with 10% FBS. For the assay, cells were plated inBiocoated 96-well microplates and incubated at 37° C. in 10% FBS(charcoal-stripped)/BRFF-BMZERO (without androgens). After 24 h, thecells were treated in the absence (blank) or presence of 1 nM DHT(control) or with test compounds (sample) of the present invention inconcentrations ranging from 10⁻¹⁰ to 10⁻⁵ M. Duplicates were used foreach sample. The compound dilutions were performed on a Biomek 2000laboratory work station. Seventy-two h later 0.44 uCi. of [³H]-Thymidine(Amersham) was added per well and incubated for another 24 h followed bytripsinization, harvesting of the cells onto GF/B filters. Micro-scintPS were added to the filters before counting them on a Beckman TopCount.

The % Inhibition was calculated as:%Inhibition=100×(1−[average_(control)−average_(blank)/average_(sample)−average_(blank)])Data was plotted and the concentration of compound that inhibited 50% ofthe [³H]-Thymidine incorporation was quantified (IC₅₀).Murine Breast Cell Proliferation Assay

The ability of compounds of the present invention (“test compounds”) tomodulate the function of the AR was determined by testing said compoundsin a proliferation assay using the androgen responsive murine breastcell line derived from the Shionogi tumor, Hiraoka et al., Cancer Res.,47, 6560-6564 (1987). Stable AR dependent clones of the parentalShionogi line were established by passing tumor fragments under thegeneral procedures originally described in Tetuo, et. al., CancerResearch 25, 1168-1175 (1965). From the above procedure, one stableline, SC114, was isolated, characterized and utilized for the testing ofexample compounds. SC114 cells were incubated with or without the testcompounds for 72 h and the amount of [3H]-thymidine incorporated intoDNA was quantified as a surrogate endpoint to assess the number of cellsand therefore the proliferation rate as described in Suzuki et. al., J.Steroid Biochem. Mol. Biol. 37, 559-567 (1990). The SC114 cell line wasmaintained in MEM containing 10⁻⁸ M testosterone and 2% DCC-treated FCS.For the assay, cells were plated in 96-well microplates in themaintenance media and incubated at 37° C. On the following day, themedium was changed to serum free medium [Ham's F-12:MEM (1;1, v/v)containing 0.1% BSA] with (antagonist mode) or without (agonist mode)10⁻⁸ M testosterone and the test compounds of the present invention inconcentrations ranging from 10⁻¹⁰ to 10⁻⁵ M. Duplicates were used foreach sample. The compound dilutions were performed on a Biomek 2000laboratory work station. Seventy two h later 0.44 uCi of [3H]-Thymidine(Amersham) was added per well and incubated for another 2 h followed bytripsinization, and harvesting of the cells onto GF/B filters.Micro-scint PS were added to the filters before counting them on aBeckman TopCount.

For the antagonist mode, the % Inhibition was calculated as:%Inhibition=100×(1−[average_(sample)−average_(blank)/average_(control)−average_(blank)])Data was plotted and the concentration of compound that inhibited 50% ofthe [³H]-Thymidine incorporation was quantified (IC₅₀).

For the agonist mode % Control was referred as the effect of the testedcompound compared to the maximal effect observed with the naturalhormone, in this case DHT, and was calculated as:%Control=100×(average_(sample)−average_(blank))/(average_(control)−average_(blank))Data was plotted and the concentration of compound that inhibited 50% ofthe [³H]-Thymidine incorporation was quantified (EC₅₀).In Vitro Assay to Measure GR-Induced AP-1 Transrepression

The AP-1 assay is a cell-based luciferase reporter assay. A549 cells,which contain endogenous glucocorticoid receptor, were stablytransfected with an AP-1 DNA binding site attached to the luciferasegene. Cells are then grown in RPMI+10% fetal calf serum(charcoal-treated)+Penicillin/Streptomycin with 0.5 mg/mL geneticin.Cells are plated the day before the assay at approximately 40000cells/well. On assay day, the media is removed by aspiration and 20 μLassay buffer (RPMI without phenol red+10% FCS(charcoal-treated)+Pen/Strep) is added to each well. At this pointeither 20 μL assay buffer (control experiments), the compounds of thepresent invention (“test compounds”) (dissolved in DMSO and added atvarying concentrations) or dexamethasome (100 nM in DMSO, positivecontrol) are added to each well. The plates are then pre-incubated for15 min at 37° C., followed by stimulation of the cells with 10 ng/mLPMA. The plates are then incubated for 7 h at 37° C. after which 40 μLluciferase substrate reagent is added to each well. Activity is measuredby analysis in a luminometer as compared to control experiments treatedwith buffer or dexamethasome. Activity is designated as % inhibition ofthe reporter system as compared to the buffer control with 10 ng/mL PMAalone. The control, dexamethasone, at a concentration of ≦10 μMtypically suppresses activity by 65%. Test compounds which demonstratean inhibition of PMA induction of 50% or greater at a concentration oftest compound of ≦10 μM are deemed active.

In Vivo Assays

Levator Ani & Wet Prostate Weight Assay AR Agonist Assay

The activity of compounds of the present invention as AR agonists wasinvestigated in an immature male rat model, a recognized test ofanabolic effects in muscle and sustaining effects in sex organs for agiven compound, as described in L. G. Hershberger et al., Proc. Soc.Expt. Biol. Med., 83, 175 (1953); B. L. Beyler et al, “Methods forevaluating anabolic and catabolic agents in laboratory animals”, J.Amer. Med. Women's Ass., 23, 708 (1968); H. Fukuda et al.,“Investigations of the levator ani muscle as an anabolic steroid assay”,Nago Dai. Yak. Ken. Nem. 14, 84 (1966) the disclosures of which areherein incorporated by reference.

The basis of this assay lies in the well-defined action of androgenicagents on the maintenance and growth of muscle tissues and sexualaccessory organs in animals and man. Androgenic steroids, such astestosterone (T), have been well characterized for their ability tomaintain muscle mass. Treatment of animals or humans after castrationswith an exogenous source of T results in a reversal of muscular atrophy.The effects of T on muscular atrophy in the rat levator ani muscle havebeen well characterized. M. Masuoka et al., “Constant cell population innormal, testosterone deprived and testosterone stimulated levator animuscles” Am. J. Anat. 119, 263 (1966); Z. Gori et al., “Testosteronehypertrophy of levator ani muscle of castrated rats. I. Quantitativedata” Boll.—Soc. Ital. Biol. Sper. 42, 1596 (1966); Z. Gori et al.,“Testosterone hypertrophy of levator ani muscle of castrated rats. II.Electron-microscopic observations” Boll.—Soc. Ital. Biol. Sper. 42, 1600(1966); A. Boris et al., Steroids 15, 61 (1970). As described above, theeffects of androgens on maintenance of male sexual accessory organs,such as the prostate and seminal vesicles, is well described. Castrationresults in rapid involution and atrophy of the prostate and seminalvesicles. This effect can be reversed by exogenous addition ofandrogens. Since both the levator ani muscle and the male sex organs arethe tissues most responsive to the effects of androgenic agents, thismodel is used to determine the androgen dependent reversal of atrophy inthe levator ani muscle and the sex accessory organs in immaturecastrated rats. Sexually mature rats (200-250 g, 6-8 weeks-old,Sprague-Dawley, Harlan) were acquired castrated from the vendor(Taconic). The rats were divided into groups and treated daily for 7 to14 days with one of the following:

1. Control vehicle

2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous)

3. TP plus Bicalutamide (administered p.o. in PEGTW, QD), a recognizedantiandrogen, as a reference compound.

4. To demonstrate antagonist activity, a compound of the presentinvention (“test compound”) was administered (p.o. in PEGTW, QD) with TP(s.c. as administered in group 2) in a range of doses.

5. To demonstrate agonist activity a compound of the present invention(“test compound”) was administered alone (p.o. in PEGTW, QD) in a rangeof doses.

At the end of the 7-14-day treatment, the animals were sacrificed bycarbon dioxide, and the levator ani, seminal vesicle and ventralprostate weighed. To compare data from different experiments, thelevator ani muscle and sexual organ weights were first standardized asmg per 100 g of body weight, and the increase in organ weight induced byTP was considered as the maximum increase (100%). Super-anova (onefactor) was used for statistical analysis.

The gain and loss of sexual organ weight reflect the changes of the cellnumber (DNA content) and cell mass (protein content), depending upon theserum androgen concentration. See Y. Okuda et al., J. Urol., 145,188-191 (1991), the disclosure of which is herein incorporated byreference. Therefore, measurement of organ wet weight is sufficient toindicate the bioactivity of androgens and androgen antagonist. Inimmature castrated rats, replacement of exogenous androgens increaseslevator ani, seminal vesicles (SV) and prostate in a dose dependentmanner.

The maximum increase in organ weight was 4 to 5-fold when dosing 3mg/rat/day of testosterone (T) or 1 mg/rat/day of testosteronepropionate (TP) for 3 days. The EC₅₀ of T and TP were about 1 mg and0.03 mg, respectively. The increase in the weight of the VP and SV alsocorrelated with the increase in the serum T and DHT concentration.Although administration of T showed 5-times higher serum concentrationsof T and DHT at 2 h after subcutaneous injection than that of TP,thereafter, these high levels declined very rapidly. In contrast, theserum concentrations of T and DHT in TP-treated animals were fairlyconsistent during the 24 h, and therefore, TP showed about 10-30-foldhigher potency than free T.

EXAMPLES

The following examples serve to better illustrate, but not limit, someof the preferred embodiments of the invention.

Examples 1 and 22-Chloro-4-((3aR,4R,6aS)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-methylbenzonitrileand2-Chloro-4-((3aS,4S,6aR)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-methylbenzonitrile

Examples 3 and 42-Chloro-4-((3aS,4R,6aS)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]pyrrol-1(2H)-yl)-3-methylbenzonitrileand2-Chloro-4-((3aR,4S,6aR)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]pyrrol-1(2H)-yl)-3-methylbenzonitrile

1A. 3-Chloro-2-methylphenylacetamide

To a solution of 3-chloro-2-methylaniline (3.0 g, 21.2 mmol) in 25 mL ofEtOH at rt was added acetic anhydride (2.4 mL, 25.4 mmol), and thesolution was stirred at rt for 2 h. The mixture was concentrated underreduced pressure to give 3.89 g (100%) of the desired acetamide. ¹H NMR(DMSO-d₆) δ 2.05 (s, 3H), 2.20 (s, 3H), 7.16 (t, J=7.7, 8.3, 1H), 7.25(d, J=8.3, 1H), 7.31 (d, J=8.3, 1H), 9.55 (s, 1H); ¹³C NMR (DMSO-d₆) δ15.1, 23.1, 124.4, 125.8, 126.7, 130.3, 133.7, 138.0, 168.3; HPLC a)column: Phenominex ODS C18 4.6×50 mm, 4 min gradient, 10% MeOH/90%H₂O/0.1% TFA to 90% MeOH/10% H₂O/0.1% TFA; 1 min hold, 4 mL/min UVdetection at 220 nm, 2.32 min retention time; HPLC b) column: ShimadzuShim-Pack VP-ODS C18 4.6×50 mm, 4 min gradient, 10% MeOH/90% H₂O/0.1%TFA to 90% MeOH/10% H₂O/0.1% TFA, 1 min hold; 4 mL/min, UV detection at220 nm, 2.20 min retention time (99%); MS (ES) m/z 184 [M+H]⁺.

1B. 4-Bromo-3-chloro-2-methylphenylacetamide

To a suspension of acetamide 1A (2.0 g, 10.9 mmol) in 15 mL of glacialAcOH cooled to approximately 15° C. was added bromine (1.67 mL, 32.7mmol) over 20 min. The ice bath was removed and the solution was stirredfor 2 h, poured into ice water with stirring, and the solid was thenfiltered and dried to give 2.75 g (96%) of the desired bromide. ¹H NMR(DMSO-d₆) δ 2.05 (s, 3H), 2.28 (s, 3H), 7.29 (d, J=8.3, 1H), 7.56 (d,J=8.8, 1H), 9.60 (s, 1H); ¹³C NMR (DMSO-d₆) δ 16.7, 23.1, 118.1, 125.5,130.4, 132.7, 133.4, 137.1, 168.4; HPLC a) column: Phenominex ODS C184.6×50 mm, 4 min gradient, 10% MeOH/90% H₂O/0.1% TFA to 90% MeOH/10%H₂O/0.1% TFA, 1 min hold, 4 mL/min, UV detection at 220 nm, 2.95 minretention time; HPLC b) column: Shimadzu Shim-Pack VP-ODS C18 4.6×50 mm,4 min gradient, 10% MeOH/90% H₂O/0.1% TFA to 90% MeOH/10% H₂O/0.1% TFA,1 min hold, 4 mL/min, UV detection at 220 nm, 2.87 min retention time(98%); MS (ES) m/z 263 [M+H]⁺.

1C. 3-Chloro-4-cyano-2-methylphenylacetamide

A suspension of bromide 1B (2.7 g, 10.3 mmol) and copper cyanide (0.92g, 10.3 mmol) in DMF (30 mL) was heated to 150° C. for 4 h. Thesuspension was cooled, poured into water with stirring, and the solidwas filtered and dried to give 1.44 g (67%) of the desired nitrile. ¹HNMR (DMSO-d₆) δ 2.12 (s, 3H), 2.29 (s, 3H), 7.72 (d, J=8.8, 1H), 7.75(d, J=8.2, 1H), 9.73 (s, 1H); ¹³C NMR (DMSO-d₆) δ 15.3, 23.5, 107.7,116.5, 123.0, 130.1, 131.5, 135.7, 142.3, 168.8; HPLC a) column:Phenominex ODS C18 4.6×50 mm, 4 min gradient, 10% MeOH/90% H₂O/0.1% TFAto 90% MeOH/10% H₂O/0.1% TFA, 1 min hold, 4 mL/min, UV detection at 220nm, 2.23 min retention time; HPLC b) column: Shimadzu Shim-Pack VP-ODSC18 4.6×50 mm, 4 min gradient, 10% MeOH/90% H₂O/0.1% TFA to 90% MeOH/10%H₂O/0.1% TFA, 1 min hold, 4 mL/min, UV detection at 220 nm, 2.13 minretention time (95%); MS (ES) m/z 209 [M+H]⁺.

1D. 3-Chloro-4-cyano-2-methylphenylaniline

A solution of cyanoacetamide 1C (9.90 g, 47.4 mmol) in 100 mL ofconcentrated HCl/EtOH (1:1) was refluxed 30 min. The solution was thenconcentrated and dried under reduced pressure to give 9.41 g (98%) ofthe desired aniline as the hydrochloride salt. The free base of theaniline was obtained by suspending the salt in EtOAc and washing withsaturated aqueous NaHCO₃ solution. The organic layer was then dried(MgSO₄), filtered and concentrated under reduced pressure. ¹H NMR(DMSO-d₆) δ 2.12 (s, 3H), 6.30 (s, 2H), 6.61 (d, J=8.23, 1H), 7.36 (d,J=8.23, 1H); ¹³C NMR (DMSO-d₆) δ 13.8, 96.9, 112.1, 118.3, 118.85,132.2, 135.6, 152.5; HPLC a) column: Phenominex ODS C18 4.6×50 mm, 4 mingradient, 10% MeOH/90% H₂O/0.1% TFA to 90% MeOH/10% H₂O/0.1% TFA, 1 minhold, 4 mL/min, UV detection at 220 nm, 2.43 min retention time; HPLCb):column: Shimadzu Shim-Pack VP-ODS C18 4.6×50 mm, 4 min gradient, 10%MeOH/90% H₂O/0.1% TFA to 90% MeOH/10% H₂O/0.1% TFA, 1 min hold, 4mL/min, UV detection at 220 nm, 2.31 min retention time (99%); MS (ES)m/z 167 [M+H]⁺.

1E. 2-Chloro-4-Iodo-3-methylbenzonitrile

To a suspension of CuI (7.5 g, 39.3 mmol) in acetonitrile (150 mL) underN₂ at rt was added tert-butylnitrite (5.7 mL, 47.9 mmol). The reactionmixture was heated to 65° C. for 1 h and then compound 1D (6.0 g, 36.0mmol) was added and the reaction was heated at 65° C. for 3 h. Thereaction was cooled to rt and filtered through a pad of celite. Thecelite pad was washed with EtOAc. The organics were washed twice withwater, dried over MgSO₄, filtered and concentrated. Purification byflash chromatography (silica gel, 110 g ISCO, 0-5% EtOAc in hexane, stepgradient) gave the title compound (4.3 g): MS (ES) m/z 278 [M+H]⁺.

1F. 6,6-Diethoxybicyclo[3.2.0]heptan-2-one

To a solution of cyclopent-2-enone (3.7 g, 45 mmol) in pentane (80 mL)was added 1,1-diethoxyethene (89 mL, 677 mmol). The mixture was put inan ACE Photo Reactor (450 W lamp), and stirred at RT for 20 hr. It wasconcentrated to give the title compound as a pale yellow oil.

1G. 2-Hydroxybicyclo[3.2.0]heptan-6-one

To a stirred solution of compound 1F in dioxane (50 mL) and 2M KH₂PO₄(50 mL) was added NaBH₄ (1.0 g, 26.4 mmol) over a 10 min-period, and thestirring was continued at RT for 1 hr. The reaction mixture was dilutedwith 2M HCl to adjust pH to 1, and stirred at RT for another 1 hr. Thereaction was concentrated, dissolved in EtOAc, washed with water, driedover Na₂SO₄, and concentrated. Purification by silica gel chromatography(50% EtOAc in hexanes) provided the title compound (1.9 g) as a paleyellow oil.

1H. 6-Oxobicyclo[3.2.0]heptan-2-yl benzoate

To a stirred solution of compound 1G (1.9 g, 15.1 mmol) in CH₂Cl₂ (10mL) at 0° C. under nitrogen were added Et₃N (3.1 mL, 22.2 mmol) andbenzoyl chloride (2.33 g, 16.6 mmol). The reaction was stirred at 0° C.for 60 min and RT for 30 min, then quenched with water. The product wasextracted with CH₂Cl₂, the organics were washed with saturated aqueousNaHCO₃, dried over Na₂SO₄, and concentrated. Purification by silica gelchromatography (5% EtOAc in hexanes) provide the title compound (3 g).

1I. 1-Oxo-octahydrocyclopenta[c]pyrrol-4-yl benzoate and

1J. 2-Oxo-octahydrocyclopenta[b]pyrrol-4-yl benzoate

To a stirred solution of compound 1H (500 mg, 2.2 mmol) in CH₂Cl₂ (5 mL)was added a solution of O-mesitylenesulfonylhydroxylamine (650 mg, 3mmol) in CH₂Cl₂ (2 mL) at −10° C., and the stirring was continued for 20min between −10 to 0° C. It was concentrated, added to a mixed solventof benzene and methanol (30 mL, 3:1) followed by a slurry of basicalumina (25 g) in methanol (20 mL). The reaction mixture was stirred atRT overnight, and alumina solid was removed by filtration. Purificationby silica gel chromatography (5% to 100% EtOAc in hexanes, then 5%methanol in EtOAc) provide mixtures of the title compounds 1I and 1J(total 360 mg) as a yellow oil. MS (ES) m/z 246 [M+H]⁺.

1K.2-(3-Chloro-4-cyano-2-methylphenyl)-1-oxo-octahydrocyclopenta[c]-pyrrol-4-ylbenzoate and

1L.1-(3-Chloro-4-cyano-2-methylphenyl)-2-oxo-octahydrocyclopenta[b]-pyrrol-4-ylbenzoate

To a vial containing mixtures of compound 1I and 1J (100 mg, 0.4 mmol)obtained from previous step in dioxane (1 mL) were added CuI (15.6 mg,0.08 mmol), K₃PO₄ (174 mg, 0.82 mmol), 1,2-cyclohexyldiamine (4.7 mg,0.08 mmol) and compound 1E (136 mg, 0.49 mmol). The vial was sealed, andheated at 110° C. for 14 hr, cooled to RT. The solid was filtered off,and filtrate was concentrated. Purification by silica gel chromatography(5% to 100% EtOAc in hexanes, then 10% methanol in EtOAc) provide themixtures of title compounds 1K and 1L (total 98 mg) as a yellow oil. MS(ES) m/z 395 [M+H]⁺.

Chiral Separation of Compound 1K and 1L

The enantiomer 1K-1 and 1K-2 were separated from the mixtures ofcompound 1K and 1L obtained in previous step. The chiral separation wasdone on a Chiralpak AD column (20% IPA in heptane) and followed byChiralpak OD (6% MeOH and 6% EtOH in heptane) to give 24 mg each.

The enantiomer IL-1 and 1L-2 were separated from the mixtures ofcompound 1K and 1L obtained in previous step. The chiral separation wasdone on a Chiralpak AD column (20% IPA in heptane) and followed byChiralpak OD (6% MeOH and 6% EtOH in heptane) to give 10 mg each.

2-Chloro-4-((3aR,4R,6aS)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-methylbenzonitrile(Example 1)

To a stirred solution of compound 1K-1 (24 mg, 0.06 mmol) in 2 mL of THFwas added KOH (90 μL, 1M in methanol) at 0° C., and the stirring wascontinued at 0° C. for 3 hr. It was quenched with 1N HCl (90 μL), anddiluted with EtOAc and water. The organic layer was separated, driedover Na₂SO₄, and concentrated. Purification by silica gel chromatography(100% CH₂Cl₂ to 5% MeOH in CH₂Cl₂ or 5%-80% EtOAc in hexanes) providethe title compound. MS (ES) m/z 291 [M+H]⁺; [α]=−80° (589 nm, c=0.091 inMeOH);

2-Chloro-4-((3aS,4S,6aR)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-methylbenzonitrile(Example 2)

The title compound was prepared from compound 1K-2 in a similar manneras for example 1 (last step). MS (ES) m/z 291 [M+H]⁺; [α]=+80° (589 nm,c=0.091 in MeOH).

2-Chloro-4-((3aS,4R,6aS)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]pyrrol-1(2H)-yl)-3-methylbenzonitrile(Example 3)

The title compound was prepared from compound 1L-1 in a similar manneras for example 1 (last step). MS (ES) m/z 291 [M+H]⁺; [α]=+13.2° (589nm, c=0.092 in MeOH).

2-Chloro-4-((3aR,4S,6aR)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]-pyrrol-1(2H)-yl)-3-methylbenzonitrile(Example 4)

The title compound was prepared from compound 1L-2 in a similar manneras for example 1 (last step). MS (ES) m/z 291 [M+H]⁺; [α]=−13.5° (589nm, c=0.092 in MeOH).

Example 52-Chloro-4-((3aR,4S,6aS)-4-hydroxy-1-oxo-hexahydrocyclopenta[e]pyrrol-2(1H)-yl)-3-methylbenzonitrile

5A.(3aR,4S,6aS)-2-(3-Chloro-4-cyano-2-methylphenyl)-1-oxo-octahydro-cyclopenta-[c]pyrrol-4-ylbenzoate

To a solution of example 1 (10 mg, 0.034 mmol) in 1 mL of THF were addedtriphenylphosphine (18 mg, 0.069 mmol), benzoic acid (8.4 mg, 0.069mmol) and diisopropyl azodicarboxylate (13.9 mg, 0.069 mmol), andstirred at RT for 3 hr. It was concentracted, and purified by prepHPLCto give the title compound (11 mg). MS (ES) m/z 395 [M+H]⁺.

5B.2-Chloro-4-((3aR,4S,6aS)-4-hydroxy-1-oxo-hexahydro-cyclopenta[c]pyrrol-2(1H)-yl)-3-methylbenzonitrile(example 5)

The title compound was prepared from compound 5A in a similar manner asfor Example 1 (last step). MS (ES) m/z 291 [M+H].

Example 62-Chloro-4-((3aS,4R,6aR)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-methylbenzonitrile

The title compound was prepared from the final compound obtained fromexample 2 in a similar manner as for example 5. MS (ES) m/z 291 [M+H].

Examples 7 and 82-Chloro-4-(((3aR,4R,6aS)-4-hydroxy-1-oxo-hexahydrocyclopenta-[c]pyrrol-2(1H)-yl)methyl)-3-methylbenzonitrileand2-Chloro-4-(((3aS,4S,6&R)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]pyrrol-2(1H)-yl)methyl)-3-methylbenzonitrile

Examples 9 and 106-Chloro-3-(((3aS,4R,6aS)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]-pyrrol-1(2H)-yl)methyl)-2-methylbenzonitrileand6-Chloro-3-(((3aR,4S,6aR)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]pyrrol-1(2H)-yl)methyl)-2-methylbenzonitrile

7A. Methyl 3-chloro-4-cyano-2-methylbenzoate

To a solution of compound 1E (5 g, 14.4 mmol) in DMF (80 mL) and MeOH(60 mL) were added 1,3-bis(diphenylphosphino)propane (1.78 g, 4.3 mmol),palladium acetate (968 mg, 4.3 mmol) and DBU (3.28 g, 21.6 mmol). Theflask was flushed with carbon monoxide (CO) several times, then heatedat 80° C. overnight under a CO balloon. The catalyst was filtered, andthe filtrate was concentrated. The residue was dissolved in EtOAc,washed with water, dried over Na₂SO₄, and concentrated. Purification bysilica gel chromatography (0-10% EtOAc in hexanes) provide the titlecompound (2.75 g) as a light pink powder.

7B. 2-Chloro-4-(hydroxymethyl)-3-methylbenzonitrile

To a pressure reaction flask containing compound 7A (1.5 g, 7.2 mmol) inTHF (30 mL) were added 1,2-ethanedithiol (2.4 g, 28.6 mmol) and NaBH(812 mg, 21.5 mmol). It was sealed, and heated at 70° C. for 2 hr, andcooled to RT. The reaction mixture was concentrated, dissolved in EtOAc,washed with water, dried over Na₂SO₄, and concentrated.Recrystallization in EtOAc/hexanes give the title compound (870 mg).(ES) m/z 182 [M+H]⁺; 204 [M+Na]⁺.

7C. 4-(Bromomethyl)-2-chloro-3-methylbenzonitrile

To a solution of compound 7B (700 mg, 3.9 mmol) in CH₂Cl₂ (15 mL) wasadded PBr₃ (5.8 mL, 5.8 mmol, 1N in CH₂Cl₂) at −15° C., and stirred at−15° C. for 3 hr. The reaction was slowly quenched with saturated NaHCO₃solution at −15° C. The organic layer was separated, dried over MgSO₄,and concentrated. Purification by silica gel chromatography (0-10% EtOAcin hexanes) provide the title compound (270 mg) as a white powder.

7D.2-(3-Chloro-4-cyano-2-methylbenzyl)-1-oxo-octahydrocyclopenta[c]-pyrrol-4-ylbenzoate and

7E.1-(3-Chloro-4-cyano-2-methylbenzyl)-2-oxo-octahydrocyclopenta[b]-pyrrol-4-ylbenzoate

To a solution containing mixtures of compound 1I and 1J (170 mg, 0.69mmol, different batch than previous lot in example 1) in anhydrous THF(4 mL) and anhydrous DMF (4 mL) was added sodium hydride (27.7 mg, 0.69mmol) at 0° C., and stirred at 0° C. for 45 min. Compound 7C (169 mg,0.69 mmol) was added at 0° C., the ice-water bath was removed, and thestirring was continued between 0° C. to RT for 2 hr before it wasquenched with saturated NH₄Cl solution. The mixture was diluted withEtOAc, organic layer was separated, washed with water, dried over MgSO₄,and concentrated. Purification by silica gel chromatography (30-100%EtOAc in hexanes) provide the title compound 7D (90 mg) and 7E (151 mg).Compound 7D: (ES) m/z 409 [M+H]⁺. Compound 7E: (ES) m/z 409 [M+H]⁺.

Chiral Separation of Compounds 7D and 7E

Both separations were done on a Chiralpak OD prep column (7.5% MeOH,7.5% EtOH in heptane) to give 45 mg each of 7D1 and 7D2; 75 mg each of7E1 and 7E2.

2-Chloro-4-(((3aR,4R,6aS)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]-pyrrol-2(1H)-yl)methyl)-3-methylbenzonitrile(Example 7)

The title compound was prepared from compound 7D1 in a similar manner asfor example 1 (last step). MS (ES) m/z 305 [M+H]; [α]=−19.0° (589 nm,c=0.080 in CH₂Cl₂).

2-Chloro-4-(((3aS,4S,6aR)-4-hydroxy-1-oxo-hexahydrocyclopenta[c]-pyrrol-2(1H)-yl)methyl)-3-methylbenzonitrile

The title compound was prepared from compound 7D2 in a similar manner asfor example 1 (last step). MS (ES) m/z 305 [M+H]; [α]=+21.3 (589 nm,c=0.081 in CH₂Cl₂).

6-Chloro-3-(((3aS,4R,6aS)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]-pyrrol-1(2H)-yl)methyl)-2-methylbenzonitrile(Example 9)

The title compound was prepared from compound 7E1 in a similar manner asfor example 1 (last step). MS (ES) m/z 305 [M+H]; [α]=−24.5° (589 nm,c=0.087 in CH₂Cl₂).

6-Chloro-3-(((3aR,4S,6aR)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]-pyrrol-1(2H)-yl)methyl)-2-methylbenzonitrile(Example 10)

The title compound was prepared from compound 7D2 in a similar manner asfor example 1 (last step). MS (ES) m/z 305 [M+H]; [α]=+24.2° (589 nm,c=0.086 in CH₂Cl₂).

1. A compound according to formula I:

or a pharmaceutically acceptable salt thereof, wherein R₁ is H; one R₂ at the carbon adjacent to the carbon to which R₁ is attached is OH and the other R₂'s are each H; R₅ is H; X is

(where N is attached to the bridgehead carbon); R₆ is selected from the group consisting of G and CH₂G; G is aryl, wherein said aryl is mono- or polycyclic, and is optionally substituted with one or more substituents selected from the group consisting of hydrogen, halo, CN, alkyl, and substituted alkyl; and n is
 1. 2. The compound or salt according to claim 1, where R₆ is 4-chloro-3-cyano-2-methylphenylmethyl.
 3. The compound or salt according to claim 2, which is


4. The compound or salt according to claim 2 which is


5. A pharmaceutical composition, comprising: at least one compound or salt according to formula I of claim 1; and at least one pharmaceutically acceptable diluent or carrier.
 6. The compound according to claim 1, wherein G is:

wherein R₈, R₉, R₁₀ and R₁₁ are independently selected from the group consisting of H, CN, halo, alkyl, and substituted alkyl; E and F are independently selected from the group consisting of N and CR₁₂; R₂ is independently selected from the group consisting of a direct bond and R₁₃; R₁₃ is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, arylalkyl, substituted arylalkyl, CO₂R₄, CONR₄R₄ and CH₂OR₄.
 7. The compound according to claim 6, wherein R₈ is CN.
 8. A compound selected from the group consisting of: 2-Chloro-4-((3aS,4R ,6aS)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]pyrrol-1(2H)-yl)-3-methylbeuzonitrile; and 2-Chloro-4-((3aR,4S,6aR)-4-hydroxy-2-oxo-hexahydrocyclopenta[b]pyrrol-1(2H)-yl)-3-methylbenzonitrile. 